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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
  • C12Q1/701—Specific hybridization probes
  • C12Q1/702—Specific hybridization probes for retroviruses
  • C12Q1/703—Viruses associated with AIDS

Definitions

• This invention relates to methods for detecting viral genotypes, and in particular Hepatitis C virus (HCV) genotypes.
• HCV Hepatitis C virus
• the hepatitis C virus is a positively stranded RNA virus that exists in six genetically distinct genotypes. These are designated Type 1, 2, 3, 4, 5 and 6, and their full length genomes have been reported (Genbank EMBL accession numbers Type la: M62321 (Choo), AF009606, AF011753, Type lb: AF054250, D13558, L38318, U45476, D85516; Type 2b: D10988; Type 2c: D50409; Type 3a: AF046866; Type 3b: D49374; Type 4: WC-G6, WC-G11, WG29 (Li-Zhe Xu et al, J.Gen. Virol.
• HCV infection affects approximately 0.5% of the population in the United Kingdom, 1.8% of the population in the USA and 3% of the population of the world. In over 85% of infected people, HCV causes a lifelong infection characterised by chronic hepatitis that varies in severity between individuals.
• interferon alpha interferon alpha
• combination therapy is highly effective in the treatment of HCV infection in patients previously treated with interferon alone and in patients never previously treated with interferon (Davis et al, NEJM, (1998), 339(21): 1493-99; Poynard et al, Lancet (1998) 352(9138): 1426-32).
• Subgroup analysis has revealed that the major determinant of prognosis and response to treatment is HCV genotype (Poynard etal, Lancet, 1998, 352(9138): 1426-32).
• genotype 1 predominates.
• Combination therapy is expensive (in the UK, it costs approximately £8,000 for one year's treatment).
• identification of patients with non-genotype 1 infections allows treatment to be stopped after 6 months with a consequent cost saving, and the avoidance of unnecessary adverse effects of the drugs associated with further treatment.
• the most serious outcome of HCV infection is heptocellular carcinoma. It is therefore desirable to be able to identify with which genotype(s) of HCV a patient is infected.
• Known HCV genotyping methods rely on agarose gel electrophoresis of restriction endonuclease digested polymerase chain reaction amplification products (RF P) or antibody detection of anti-HCV antibodies. Enzymatic digestion can take a relatively long time to reach completion making the RFLP method time consuming. It also involves several different stages, making it unamenable to automation or rapid throughput. In addition, inaccurate genotyping can occur owing to (i) the fact that restriction endonucleases do not cut with 100% efficiency, and (ii) single base pair mutations in quasispecies may result in failure of digestion of the PCR products. Antibody based methods are costly and relatively insensitive. Most significantly, they are incapable of differentiating between patients who have cleared HCV infection and those with persisting infection.
• US Patent No. 5851759 discloses methods of genotyping HCV in which HCV RNA is isolated and cDNA is synthesised from this RNA. The cDNA is then subjected to PCR using primers which flank the El region of the HCV genome. The products of PCR are analysed using the Heteroduplex Traching Assay (HTA). The El region was selected for analysis because it is considered to be the most heterogeneous region of the HCV genome. Thus, amplification of this region is likely to give a different product for each HCV strain in a sample. These products can then be analysed to indicate the strains that are present in the sample.
• HTA Heteroduplex Traching Assay
• HCV genotype 1 HCV-1
• a method of detecting HCV genotype 1 (HCV-1) in a sample comprising: subjecting the sample to an amplification reaction using at least one primer which anneals specifically to the 5' noncoding region (5' NCR) of the HCV-1 genome; and, detecting the product of the amplification reaction.
• the HCV genome consists of 5' and 3' noncoding (or untranslated) regions (NCR) that flank a single long open reading frame (ORF).
• This ORF encodes for three structural proteins at the amino-terminal end and for six non-structural (NS) proteins at the carboxyl-terminal end.
• the structural proteins are represented from the nucleocapsid (core; C) proteins and two glycoproteins, envelope 1 (El) and envelope 2 (E2).
• the non-structural proteins are named NS2, P7, NS3, NS4a, NS4b, NS5a, NS5b.
• the 5' NCR is the most highly conserved part of the HCV genome, whereas the sequence of the two envelope proteins (El and E2) is highly variable among different HCV isolates.
• the present invention lies in the surprising finding that parts of the 5' NCR are conserved between HCV-1 quasi species but not between other genotypes.
• a preferred region is the region between residues -134 and -118 of the 5'NCR of HCV- 1. Accordingly, primers for amplification reactions can be designed which anneal specifically to those parts of the 5' NCR that are conserved between HCV-1 quasi species but not between other genotypes.
• the amplification reaction may be PCR (see for example U.S. Patents Nos. 4,683,195 and 4,683,202, and rnnis et al, editors, PCR Protocols, (Academic Press, New York, 1989; Sambrook et al, Molecular Cloning, Second Edition, (Cold Spring Harbour Laboratory, New York 1989)).
• PCR will generally be used when HCV RNA has been isolated and converted, preferably by reverse transcription, to cDNA.
• PCR is carried out using Taq DNA polymerase, e.g. AmplitaqTM (Perkin-Elmer, Norwalk, Conn.).
• Taq polymerase can also be obtained from MBI Fermentas, Perkin Elmer, Boehringer Mannheim and Beckman Instruments.
• thermostable, DNA polymerase may also be used in the method of the present invention, such as Tfl (Thermus flavus) polymerase (Gut et al, Virol. Methods 77(1): 37-46 (1999)).
• the amplification reaction may be RT-PCR (Yajima et al, Clin. Chem, 44(12): 2441-2445 (1998); Martell et al, J. Clin. MicrobioL, 37(2): 327-332 (1999);
• PCR involves the extraction and denaturation (preferably by heat) of a sample of DNA (or RNA).
• a molar excess of oligonucleotide primers is added, along with a polymerase, which may be heat-stable, and dNTPs for forming the amplified sequence.
• the oligonucleotide primers are designed to hybridise to opposite ends of the sequence which it is desired be amplified.
• the polymerase replicates the DNA to produce two "long products," which begin with the respective primers.
• the total DNA which includes the two long products and the two original strands, is then denatured and a second round of polymerisation is carried out (for example, by lowering the temperature).
• the result of the second round is the two original strands, the two long products from the first round, two new long products (produced from the original strands), and two "short products" produced from the long products. These short products have the sequence of the target sequence (sense or antisense) with a primer at each end.
• the number of short products grows exponentially, each round producing two additional long products and a number of short products equal to the sum of the long and short products remaining at the end of the previous round.
• Oligonucleotide primers can be synthesised by a number of approaches, e.g. Ozaki et al, Nuc. Acids Res. 20: 5205-5214 (1992); Agrawal et al, Nuc. Acids Res. 18: 5419- 5423 (1990) or the like. Conveniently, the oligonucleotide probes are synthesised on an automated DNA synthesiser, e.g. an Applied Biosystems, Inc, Foster City, •: ⁇
• DNA/RNA synthesiser using standard chemistries such as phosphoramidite chemistry (Beaucage and Iyer, Tetrahedron 48: 2223-2311 (1992), US Patent Nos. 4980460, 4725677, 4415732, 458066 and 4973679). Alternative chemistries, including non-natural backbone groups such as phosphorothioate and phosphoramidate, may also be employed, provided that the hybridisation efficiencies of the resulting oligonucleotides are not adversely affected.
• the precise length and sequence of the DNA primers will depend on the target polynucleotide to be amplified. Preferably, the length of the DNA primers is in the range 10 to 60 nucleotides and more preferably in the range 15 to 30 or 25 nucleotides.
• the method of the present invention further comprises a step in which HCV infection generally is detected.
• the sample is subjected to an amplification reaction using primers which anneal to a region of the 5'NCR which is conserved between all HCV genotypes and the product of this amplification reaction is detected.
• the amplified region spans the region which is conserved in HCV-1. This additional step allows verification that HCV is present in the sample and so treatment can be tailored accordingly if no HCV-1 is detected.
• Detection of the products of amplification may be carried out using the well-known technique of agarose gel electrophoresis. Jf HCV-1 is present in the sample, the amplification reaction produces a product (of known size) which can be detected on the agarose gel.
• An HCV-1 specific primer suitable for this is the DNA oligonucleotide:
• Spec-1 may be used together with any universal HCV primer.
• One example is:
• the invention provides such primers alone and in combination.
• a pair of primers universal for all HCV genotypes can be used. This will produce a product of a known size which is different to that for the HCV-1-specific amplification. If HCV-1 and other HCV genotypes are present, the gel will show both products. Suitable primers for this are:
• a preliminary amplification may be carried out on the sample to isolate HCV material.
• This preliminary amplification can be carried out using two primers universal for all HCV genotypes. Suitable primers for this are: Forward: 5* GGA ACT ACT GTC TTC ACG C 3' (UTR-L1) Reverse: 5' ACG GTC TAC GAG ACC TC 3' (UTR-R1)
• Detection of the products of amplification may alternatively be carried out using fluorescent analysis. If genotype 1 HCV is present, amplification occurs and this is detected by hybridisation of a quenched fmorescently labelled probe. With successive rounds of amplification, an increasing amount of the fluorescent probe becomes incorporated into PCR products and the quencher is liberated resulting in an increasing amount of fluorescence being detectable on the reaction vessels. Such fluorescence detection makes this approach ideally suited to automation and high throughput processes.
• the oligonucleotide probe is hybridised to the target polynucleotide downstream from a primer for a polymerase having a 5'-3' exonuclease activity.
• the polymerase extends the primer as part of the PCR reaction, the oligonucleotide is digested, releasing one of the "quencher” and “reporter” molecules and causing the distance between these molecules to become such that the fluorescent emission is no longer quenched by energy transfer.
• a fluorescent signal is generated, providing realtime monitoring of amplification.
• the oligonucleotide probe may be made in the same way as the oligonucleotide primers mentioned above and may have the same variations in backbone and so on, providing that hybridisation to the target polynucleotide is not compromised and/or cleavage efficiency of the exonuclease are not adversely affected.
• the length of the oligonucleotide probe is in the range of 10 to 60 nucleotides, and more preferably 18 to 30 nucleotides.
• the precise length and sequence of the oligonucleotide probe depends at least in part on the nature of the target polynucleotide to which it binds and will be varied to achieve appropriate annealing and melting properties for a particular target polynucleotide.
• the binding location of the probe may be varied according to the nature of the target polynucleotide.
• Preferred primers for this reaction are UTR-R2 and Sped mentioned above.
• a preferred probe has the following sequence and is specific to HCV-1: .
• the technique such as the TaqMan technique can be used to detect HCV-
• the present invention provides a method of detecting HCV genotype 1 (HCV-1) in a sample, comprising: subjecting the sample to an amplification reaction using at least one primer which anneals to the genome of HCV, a polymerase having a 5 '-3' exonuclease activity and an oligonucleotide probe, which probe anneals specifically to the 5' noncoding region (5' NCR) of the HCV-1 genome and which incorporates a modified nucleotide having a fluorescent characteristic which is modified by one or more neighbouring nucleotides; and detecting a change in fluorescence as the oligonucleotide probe is degraded by the exonuclease activity of the polymerase as the polymerase extends the primer and modification of the fluorescent characteristic of the modified nucleotide is reduced.
• the modified nucleotide is preferably a "reporter” molecule and the neighbouring nucleotide(s) is/are preferably a "quencher” molecule.
• the primer(s) may be universal and is/are preferably UTR-L1 and UTR-R1 or UTR-L2 and UTR-R2.
• the probe(s) may be capable of annealing specifically to the 5' NCR of the HCN-1 genome.
• the probe may be LI as defined above.
• a further alternative method of detecting the amplification products is using one or more molecular beacon probes.
• PCR primers comprising a sequence attached to a hairpin loop sequence which contains both a fluorescent label and a quencher molecule.
• the hairpin brings the fluorescent molecule in opposition to the quencher so that the primer does not fluoresce under normal conditions.
• the hair-pin opens and the fluorescent beacon and quencher become separated. The resulting PCR product fluoresces.
• PCR primers are used:
• MBP-LR-1 is a forward HCN type 1 specific primer and, as mentioned above, UTR-R2 is a universal reverse primer. Any universal HCN primer can be used together with MBP-LR-1. If it is desired to detect the presence of other HCV genotypes in the sample, an additional amplification can be carried out using at least one molecular beacon probe which is universal for all HCV genotypes.
• a suitable probe in this regard is MBP-LR- ALL which is a forward primer and has the following sequence:
• MBP-LR-ALL can be used together with any universal reverse primer; one suitable example is UTR-R2.
• Fluorochromes that emit light at differing wavelengths can be incorporated into the different primers so that products amplified with different primers can be distinguished in the same reaction vessel.
• Alternative hairpin sequences on the beacon probes can easily be designed by the skilled person.
• the amplification products can be detected using DNA hybridisation employing enzyme linked methods to confirm hybridisation, such as using horse radish peroxidase on sequence specific probes and an appropriate substrate.
• kits for detecting HCV genotype 1 (HCV-1) in a sample comprising: at least one primer which anneals specifically to the 5' noncoding region (5' NCR) of the HCV-1 genome.
• the kit may further comprise the polymerase and an appropriate primer or set of primers.
• it may comprise additional reagents that are necessary for performing the invention, such as dNTP mixtures, buffers, molecular size standards, wax beads and the like.
• the reagents may be provided in pre-measured amounts so as to simplify the performance of the method.
• the kit may also contain detailed instructions for carrying out the method.
• sequences of the primers and probes described above can be modified without affecting their activity, i.e. their ability to act as primers or probes in the methods of the present invention.
• modified probes and primers are included in the present invention, provided that they have substantial identity with the probes mentioned above.
• BESTFTT When comparing nucleic acid sequences for the purposes of determining the degree of homology or identity, one can use programs such as BESTFTT and GAP (both from the Wisconsin Genetics Computer Group (GCG) software package).
• BESTFTT for example, compares two sequences and produces an optimal alignment of the most similar segments.
• GAP enables sequences to be aligned along their whole length and finds the - optimal alignment by inserting spaces in either sequence as appropriate.
• the comparison is made by alignment of the sequences along their whole length.
• sequences which have substantial identity have at least 75% sequence identity and more preferably at least 90 or at least 95% sequence identity with said sequences.
• sequence identity may be 99% or above.
• the term "substantial identity” indicates that said sequence has a greater degree of identity with any of the sequences described herein than with prior art nucleic acid sequences.
• Figures la and lb show 2% agarose gels of PCR products stained with ethidium bromide to show bands 88 base pairs in size (genotype 1 specific band) and 217 base pairs (all HCV genotypes).
• Figures 2a-f show printouts of PCR runs using fluorescent probe detection and Taqman technology. Fluorescence intensity is represented on the y axis and the number of PCR cycles elapsed is shown on the x axis.
• Figures 3a and 3b are printouts of PCR runs using fluorescent probe detection and Taqman technology. Fluorescence intensity is represented on the y axis and the number of PCR cycles elapsed is shown on the x axis.
• Figure 4 shows printouts of PCR runs using molecular beacon probe analysis and fluorescence detection. Fluorescence intensity is represented on the y axis and the number of PCR cycles elapsed is shown on the x axis.
• Figure 5 is a diagram illustrating the positions of the various primers and probes used in certain embodiments of the present invention within the 5 'NCR of the HCV-1 genome. Examples
• RNA was extracted using rnRNA extraction kit (QIAgen Ltd, Crawley, West Hampshire) as per manufacturer's instructions. 11.5 ⁇ l of RNA solution as extracted above was added to 0.5 ⁇ l of random primers (Promega UK
• cDNA was then prepared by reverse transcription using the following reaction mix:
• l ⁇ l of the resulting complimentary DNA was suspended in 20 ⁇ l of a first round reaction mix.
• This mix contains 1.5mM MgCl 2 , 0.2mM deoxy nucleotide triphosphates (guanine, adenosine, threonine and cytosine) (Amersham Pharmacia, Amersham, UK), O.l ⁇ l of Taq polymerase (Promega UK Ltd, Southampton) and 500nM each of primers UTR-L1 and UTR-R1 (from Cuachem Ltd, Glasgow).
• UTR-L1 is the forward primer and is complementary to residues -273 to -291 of the 5'UTR of HCVl. It has the sequence:
• UTR-R1 is the reverse primer and is complementary to residues -6 to -22 of the 5'UTR of HCVl. It has the sequence:
• the PCR reaction conditions were as follows; 94 C for 1 minute for 1 cycle; 94 ° C for 30 seconds; 55 C for 20 seconds; 72 C for 20 seconds for 25 cycles.
• UTR-L2 is a forward primer which is universal for all HCV genotypes and is complementary to residues -248 to -266 of the 5'UTR of HCVl. It has the following sequence:
• Spec-1 is a forward primer which is HCV type 1 specific and is complementary to residues -118 to -134 of the 5'UTR of HCVl . It has the following sequence:
• 5'CCI CTC AAT GCC TGG AG3' UTR-R2 is a reverse primer which is universal for all HCV genotypes and is complementary to residues -50 to -71 of the 5'UTR for HCVl. It has the following sequence:
• reaction conditions were as follows;94 C for 1 minute for 1 cycle; 94 C for 30 seconds; 58 C for 20 seconds; 72 C for 20 seconds for 25 cycles.
• RNA was isolated and cDNA produced in the same manner as in Example 1.
• LI is a HCV genotype 1 specific probe (prepared by Scandinavian Gene Synthesis AB, Koping, Sweden) which is complementary to residues -68 to -93 of the 5'UTR of
• HCVl has the following sequence:
• reaction conditions were as follows: 50 C for 2 minutes, 95 C for 10 minutes; for
• HCV-1 can be differentiated from the other subtypes when the primer specific for HCV-1 is used (Spec-1) (see Figures 2b, d and f).
• This approach relies on sequence specific hybridisation of a fluorescently labelled probe to genotype 1 HCV sequences. These sequences are generated by PCR using at least one primer specific for HCV-1.
• the probe has a quencher molecule linked to the fluorochrome. As PCR proceeds, the quencher molecule is removed by Taq polymerase from incorporated probes. Fluorescence of the resulting genotype 1 PCR products can then be detected. The amount of fluorescence generated during the successive rounds of PCR correlates with the amount of PCR product.
• RNA was isolated and cDNA produced in the same manner as in Example 1.
• MBP-LR-ALL is a forward primer which is universal for all HCV genotypes and is derived from the primer UTR-L2. It has the following sequence:
• MBP-LR-1 is a forward HCV type 1 specific primer which is derived from the primer Sped. It has the following sequence:

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